Composition consisting of a polymer containing amino groups and an aldehyde containing at least three aldehyde groups

ABSTRACT

The invention relates to a composition of at least two, in particular two, biocompatible components which can be chemically crosslinked together, in particular for gluing biological tissue, comprising at least the following components: a) aqueous solution of at least one polymer having amino groups b) aqueous solution of at least one aldehyde having at least three aldehyde groups, where the composition is free of protein. The invention further relates to a provision of the composition for use as surgical tissue glue, and to a kit consisting of two substantially separate containers which contain components of the composition.

The invention relates to a composition of at least two, in particulartwo, biocompatible components which can be chemically crosslinkedtogether, in particular for gluing biological tissue, comprising atleast the following components:

-   a) aqueous solution of at least one polymer having amino groups-   b) aqueous solution of at least one aldehyde having at least three    aldehyde groups.

In surgery, mainly suture materials and clips are used to join separatedportions of tissue. However, these techniques reach their limits inparticular in minimally invasive surgery, which includes inter alialaparoscopy, thoracoscopy, athroscopy, heart surgery and intraluminalendoscopy. In these areas, the use of tissue adhesives and sealants issimpler, quicker and more reliable. Several patents describe syntheticand natural polymeric or macromeric systems which can be used for gluingsoft tissues and for sealing air and fluid leaks in organs and bloodvessels.

Fibrin adhesives commercially available on the market consist inter aliaof human or/and bovine plasma proteins which represent a considerablehealth risk in relation to the transmission of infections. In addition,their adhesive force is often inadequate.

Compared with fibrin adhesives, hydrogels have distinctly greatercohesive and adhesive properties. Particularly suitable compositions arethose which can be applied in the liquid state to the tissue and thencure within a short time through the formation of covalent bonds. The insitu curing is usually based on the crosslinking of macromeric systemsand may take place by free-radical polymerization or by chemicalreaction with bi- or multifunctional crosslinking reagents.

Free-radical crosslinking can be induced by sources of light or heat,and by oxidative free-radical formation with inorganic persulfates orenzymes. U.S. Pat. No. 6,156,345, Chudzik et al., U.S. Pat. No.6,083,524, Sawhney et al. and U.S. Pat. No. 6,060,582, Hubbel et al.describe synthetic macromers with free-radical polymerizable end groupswhose polymerization is initiated by irradiation with UV light in situon the tissue. Besides synthetic polymers, it is also possible in thisway to crosslink viscous solutions of collagen and collagen derivatives(U.S. Pat. No. 6,183,498 B1, Devore et al., U.S. Pat. No. 5,874,537Kelman et al.). The technique is very elaborate and costly because ofthe additionally required source of light. As an alternative to UVactivation, polymerization can also be induced by means of a source ofheat. However, the necessary temperatures damage healthy cells in thetissue and frequently kill them. In principle, damage to healthy tissueis a problem with most free-radical polymerizations because they proceedexothermically, i.e. heat is released to the surroundings during thepolymerization.

As an alternative to free-radical polymerization, macromers can also bechemically cross-linked via reactive groups. Carbonyl reactions inparticular, as well as certain carboxyl reactions, have the desiredproperties, in terms of kinetics, to ensure rapid gelation of thecomponents. U.S. Pat. No. 6,051,648, Rhee et al., describes syntheticpolymers with N-hydroxysuccinimide activated carboxyl groups whichcrosslink with nucleophilic multifunctional polymers, with eliminationof the N-hydroxysuccinimide. Owing to the lack of stability of theactivated carboxyl groups in aqueous solution, it is necessary in thiscase to use preformed patches, which entails considerable disadvantagesin particular in minimally invasive surgery.

Free lysine units in polypeptides and proteins form Schiff's basesthrough reaction with di- or polyaldehydes. Kowanko describes in U.S.Pat. No. 5,385,606 an adhesive composition consisting of human or animalprotein and a di- or polyaldehyde, with the crosslinking preferablybeing carried out with glutaraldehyde. However, the use ofglutaraldehyde is critical. Vries et al. (Abstract Book of the SecondAnnual Meeting of the WHS, Richmond, USA p. 51, 1992) were able todemonstrate that gelatin crosslinked with glutaraldehyde had a toxiceffect on cells, which is not the case with pure gelatin.

In U.S. Pat. No. 6,156,488 by contrast, Tardy et al. describes abiocompatible tissue adhesive consisting of an aqueous collagen solutionand of an aqueous polyaldehyde solution and thus avoids the use of smalltoxic molecules for the crosslinking. A tissue adhesive composed ofoxidized dextran or starch and modified gelatin is also described by Moet al. in J. Biomater, Sci. Polymer Edn. 2000, 11, 341-351. Dextran ispresent in many medical devices and is used for example as crosslinkingcomponent in oxidized form in wound dressings (Schacht et al, U.S. Pat.No. 6,132,759). The macromolecular crosslinking reagents are in thiscase prepared by oxidizing dextran or starch with sodium periodate. Thisreaction is described inter alia by Bernstein et al. (Natl. Cancer Inst.1978, 60, 379-384) and is state of the art. The use of collagen inmedicine is, by contrast, critical in relation to the risk of infection,particularly with regard to BSE and Kreutzfeldt-Jakob diseases. Inaddition, immune responses can be induced in the body by proteins.

Chitin is in nature a widespread linear, nitrogen-containingpolysaccharide and forms the main constituent of the exoskeleton ofarthropods (cockchafer wings, lobster and shrimp shells). Chitin isconverted in concentrated sodium hydroxide solution into thedeacetylation product chitosan which, in contrast to chitin, has freeamino groups and is soluble in weakly acidic aqueous medium. Thedegradation behavior of pure and glutaraldehyde-treated chitosan, andthe acute toxicity and the hemostatic effect of chitosan is described byRao et al. (J. Biomed. Mater. Res. 1997, 34, 21-28). On the basis of theantimicrobial and hemostatic effect in combination with their highbiocompatibility, chitosan and chitin are promising substances formedical devices. In U.S. Pat. No. 6,124,273, proteins are incorporatedinto chitosan sponges, and the composition is employed for externalwounds. The chitosan sponges in this case release the proteins andexpedite wound healing. Ono et al. describe a biological tissue adhesivecomposed of photocrosslinked chitosan (K. Ono, et al., J. Biomed. Mater.Res. 2000, 49, 289-295). The crosslinking takes place by irradiationwith UV light. This costly and elaborate technique has, as alreadymentioned, not achieved practical use. In addition, the adhesive forceof this adhesive is inadequate, being in the range of the fibrinadhesives.

The invention is based on the object of producing a composition whichovercomes the prior art disadvantages mentioned, in particular avoidsthe risk of transmission of infectious diseases.

This object is achieved according to the invention by a compositionhaving the features of claim 1. Preferred embodiments and developmentsof the composition of the invention are characterized in the dependentclaims.

The fact that the composition of the invention is free of proteineliminates the risk of transmission of infectious diseases which ispresent on use of protein (e.g. collagen). This is a great advantage,especially with regard to a possible transmission of BSE pathogens tohumans, compared with the protein-containing compositions described inthe prior art. In addition, the risk of protein-related immune responsesis also excluded with the protein-free composition of the invention.

A further advantage of the composition of the invention is that thegelation of the components takes, place spontaneously, and no additionalsources of energy are required. Application is thus simplified andproceeds without harming tissues, because the healthy tissue is notadversely affected for example by excessively high heat energy.

A further advantage of the invention is that the components can beapplied in aqueous medium and thus better covering of the wound area isensured than is the case for example with preformed patches (cf., forexample, U.S. Pat. No. 6,051,648).

In a particularly preferred embodiment of the composition of theinvention, the aldehyde and the polymer having amino groups can becrosslinked together at body temperature. It is thus possible to avoidthe abovementioned additional sources of energy, which in turn avoidstissue damage.

The polymer having amino groups is preferably derived from abiodegradable natural material. It is particularly preferred for thepolymer having amino groups to be a polysaccharide, in particular amodified saccharide in which the amino groups are liberated bydeacetylation. In a particularly preferred embodiment of the compositionof the invention, the polymer having amio groups is an at leastpartially deacetylated chitin having a degree of deacetylation of from50 to 100%, preferably 60 to 90%, in particular 70 to 80%. Thedeacetylation converts the acetamide groups in the chitin into aminogroups. The effect of this in turn is, inter alia, that degradation inthe body proceeds more slowly than with (nondeacetylated) chitin. It isparticularly preferred for the polymer having amino groups to bechitosan. Chitosan has a procoagulant effect. Deacetylated chitin,especially chitosan, is preferably employed in water-soluble salt form(chloride, acetate, glutamate).

In a further embodiment of the composition of the invention, the polymerhaving amino groups is a synthetic polymer, in particular a polymerwhich undergoes renal elimination. This has the advantage that simpleexcretion of the polymer having amino groups is possible with the urine.The synthetic polymer is advantageously a modified polyvinyl alcoholhaving amino groups, preferably having a molecular weight of ≦50 000, inparticular <50 000, preferably ≦20 000, in particular <20 000.

Examples of the modification of polyvinyl alcohol are the esterificationof polyvinyl alcohol with amino acids, esterification with dicarboxylicacids or anhydrides linked to amide formation with polyfunctionalamines, especially diamines, and formation of cyclic acetals.

The degree of modification can be set at any level and is not confinedto the ends of the chains as, for example, in PEG orpolyhydroxyalkanoates. Further multifunctional polymers having freehydroxy groups which are available are also polysaccharides such asdextran, cellulose, chitosan, hyaluronic acid, alginic acid, starch,agar, chitin and chondroitin sulfate.

Examples of Modifications of Polyvinyl Alcohol (PVA)

a) Retrosynthesis of Alanine-Modified PVA

Attachment of amino acids to polyvinyl alcohol takes place in two steps.Firstly, the alcohol is esterified with a BOC-protected alanine. A baseis added as catalyst. The reaction must be carried out in anhydroussolvent. After successful attachment, the BOC protective group can beeliminated under mild conditions at room temperature withtrifluoroacetic acid. It is possible in principle for any desired aminoacid to be attached in this way, but preference is given to amino acidshaving additional thiol or hydroxy groups, such as, for example,cysteine, serine, threonine, tyrosine, with particular preference foramino acids having further amino groups, such as, for example,asparagine, lysine, glutamine, arginine or trytophan. Attachment of amixture of the amino acids mentioned is likewise conceivable.

Advantage:

-   -   no amide linkages, ester linkages ought to be cleavable by        hydrolysis    -   degradation product is an amino acid (toxicologically        acceptable)

b) Retrosynthesis with Succinic Anhydride and Diamine

The attachment of free amino groups to polyvinyl alcohol via cyclicdianhydrides likewise takes place in two stages. In the first step, theanhydride is bound to the alcohol with the aid of a catalytically actingbase EDC. This is followed by reaction with a diamine. The diamineshould be employed in excess in order to avoid crosslinking of thepolyvinyl alcohol during the reaction. Dianhydrides which can be usedare, inter alia, also maleic anhydride, adipic anhydride or glutaricanhydride.

Advantage:

-   -   Starting substances are very favorable    -   Attachment to PVA through ester linkage can easily be degraded    -   Diamines might be toxicologically problematic (possible        replacement by triethylene glycol diamine)        (NH₂—C₂H₄—O—C₂H₄—O—C₂H₄—NH₂))

c) Introduction of Terminal Amino Groups via Cyclic Acetals

Terminal amino groups can be introduced into the polyvinyl alcohol inone step via acetal linkages. The formation of the cyclic acetal isenergetically preferred in this case. The chain length of the spacer canbe varied, with preference for n≦4 and particular preference for n=1.

Advantages:

-   -   Introduction of the amino group in a single synthetic step    -   No protective group chemistry, no cross-linking is to be        expected during the reaction.

It is also possible to use combinations of polysaccharides having aminogroups and polyvinyl alcohols having amino groups.

The aldehyde is advantageously a polyaldehyde. The latter is preferablyof biological origin. In a preferred embodiment of the composition, thealdehyde is an oxidized polysaccharide. It is particularly preferred forboth the polymer having amino groups and the aldehyde to havepolysaccharide structures. In a particularly preferred embodiment of thecomposition, the aldehyde is an oxidized polysaccharide, thepolysaccharide being at least one from the group of dextran, chitin,starch, agar, cellulose, alginic acid, glycosaminoglycans, hyaluronicacid, chondroitin sulfate and derivatives thereof. Dextranaldehyde ispreferred. The aldehyde, especially the dextranaldehyde, preferably hasa molecular weight of about 60 000 to 600 000, in particular about 200000. Higher molecular weights, in particular of at least 200 000, resultin high degrees of crosslinking.

The aldehyde is advantageously partially or completely masked. Thepurpose of the masking, especially of oxidized polyaldehydes, is toprevent the formation of intermolecular acetals and thus ensure thestability of the solutions. Controlled liberation of the aldehydesfinally takes place in situ through controlled hydrolysis in a pH rangefrom 2 to 6, preferably 2 to 4.5. It is particularly preferred for thealdehyde to be masked with an S, O or N nucleophile. It is advantageousfor the partially or completely masked aldehyde to be apolysaccharide-alkali metal bisulfite adduct. In a further embodiment ofthe composition of the invention, the aldehyde is partially orcompletely masked with ethanol or glycerol.

It is advantageous for the pH values of the components to be adjusted sothat the pH of a mixture of the components is between 3 and 8, inparticular between 5 and 7.5. Although a high pH favors crosslinking, itleads to precipitation of, for example, chitosan.

The aldehyde in particular is responsible for the adhesive force andenables bonding to the tissue, but coverage of the tissue is impossiblethrough the aldehyde alone. Thus, the stoichiometric amount of aldehydegroups in component b) is advantageously at least three times thestoichiometric amount of amino groups in component a).

The components are advantageously adjusted with respect to one anotherso that they crosslink together in a short time, in particular a time offrom 15 to 200 seconds, after they are combined. The crosslinking timecan be controlled for example through the concentration of the solutionsand via the mixing ratio. The degree of crosslinking can likewise beadjusted, specifically via the number of aldehyde groups in thealdehyde.

The viscosity of the composition can also be controlled. The viscositiesof the components are advantageously adjusted in relation to one anotherso that a layer of the composition with a thickness of from 0.1 to 1 mmcan be applied.

The possibilities of adjustment which have been mentioned (e.g.crosslinking rate, viscosity, reactivity) do not apply to compositionswith gelatin or collagen, which have been modified according to theirsource and do not permit defined reactions.

The content of aldehyde of component b) is advantageously from 5 to 20%by weight, in particular from 10 to 15% by weight. The content ofpolymer having amino groups of component a) is preferably from 1 to 25%by weight, in particular from 2 to 20% by weight. The ratios by volumeof the two solutions a):b) are between 5:1 and 1:5, preferably between3:1 and 1:3. If they are 1:1, which is preferred in many cases, it ispossible in a simple manner to mix equal volumes together.

In a particularly preferred embodiment of the composition of theinvention, component a) is a solution of chitosan in acetic acid, andcomponent b) is an aqueous solution of dextranaldehyde. Dextran isdistinguished for example from glutaraldehyde (cf., for example, U.S.Pat. No. 5,385,606) by being non-toxic.

The invention additionally relates to the provision of the compositionof the invention for use as surgical adhesive, in particular for sealingor closing surfaces or orifices.

The components are preferably mixed together shortly before application.This can take place for example with the aid of a double-barrel syringein which the two components are forced into a joint ejection tube inwhich a static mixer is present. The two components are mixed togetherby the static mixer in the ejection tube and are ejected, shortly beforethey crosslink together, from the syringe onto the application site.

A further possibility is also to mix the components only on anapplication site by applying the two components for example shortly oneafter the other to an application site.

The invention also claims a kit consisting of two containers which aresubstantially separate in relation to the contents, where each containerin each case contains one component of the composition of the invention.In a preferred embodiment, the two containers function as syringebarrels of a double syringe. With a double syringe of this type, whichis also called a double-barrel syringe, the separately stored componentsare forced into a joint ejection tube. The kit advantageously has adevice for mixing the components. It is particularly preferred for thekit to have a static mixer which can be fitted in particular onto adouble syringe. This static mixer is located in particular in theejection tube of the double syringe. In a further embodiment, the doublesyringe can be closed and opened at the place where the ejection tube isfitted.

DESCRIPTION OF THE FIGURE

FIG. 1 shows a diagrammatic longitudinal section through a preferredembodiment of the kit of the invention.

The preferred embodiment of a kit of the invention which is depicted inthe single drawing shows a longitudinal section through a double syringe1. This double syringe consists of two connected syringe barrels 2 a and2 b which contain the two components of the composition of the inventionseparately. The ratios of the volumes of the two syringe barrels areadjusted to suit the mixing ratio of the two components. In the presentexemplary embodiment, the two syringe barrels 2 a and 2 b have the samevolumes of two components of a composition of the invention. It is alsopossible to use double syringes in which the volumes are different, forexample the barrels have diameters of different sizes.

The double syringe 1 additionally includes two syringe plungers 3 a and3 b which are connected together by a connecting plate 4. Two pistonsealing rings 5 a and 5 b are attached at the upper end of each of thetwo syringe plungers 3 a and 3 b. These piston sealing rings are insubstantially air-tight contact with the walls of the two syringebarrels 2 a and 2 b. At its upper end, each of the two syringe barrels 2a and 2 b have in each case a mutually directly adjoining orifice 6 a or6 b. These orifices are closed until the first use.

After the two adjoining orifices 6 a and 6 b have been opened it ispossible to fit an ejection tube 7 thereon. A static mixer 8 is locatedin the ejection tube 7. The ejection tube narrows at its upper end toform an ejection orifice 9.

The two syringe plungers 3 a and 3 b with the piston sealing rings 5 aand 5 b affixed thereon are moved, for example by pressure on theconnecting plate 4 and counter-pressure on the plate 10, in thedirection of the orifices 6 a and 6 b. This forces the two componentspresent in the syringe barrels out of the orifices 6 a and 6 b into theejection tube 7. The two components are intimately mixed together in theejection tube in particular by the static mixer 8 which is located inthe ejection tube 7, and are finally forced in the mixed state out ofthe ejection orifice 9 onto an application site.

Example of a Composition of the Invention

1. Composition

Solution A: aqueous solution of chitosan

Solution B: aqueous solution of dextranaldehyde

Mixing the two solutions results in formation of a gel which hasadhesive properties. The gelation is based on the formation of imines(Schiff's bases) between the aldehyde groups in the oxidized dextran andthe free amino groups in the chitosan (see reaction scheme approach 1).

As an alternative to chitosan solution, it is also possible to usesolutions of modified polysaccharides (dextran modified with amines) orsynthetic polymers (polyvinyl alcohol modified with amines).

1.1. Chitosan Solution

2 g of chitosan are added to 100 ml of 2% strength acetic acid solution(v/v) and stirred at room temperature for five days.

A 4% strength aqueous (w/v) Protasan® UP CI 213 (from FMC Biopolymers,Drammen, Norway) solution (deionized water) is used as alternativethereto. Protasan® UP CI 213 is a chitosan salt with chloride as counterion.

1.2 Synthesis of Dextranaldehyde

The 5% strength (w/v) sodium periodate solution used for the synthesisis freshly prepared before each reaction and is combined with a 10%strength (w/v) dextran solution. Dextranaldehydes can be prepared byusing various stoichiometric ratios (see table 1). The reaction mixtureis stirred at room temperature overnight, dialyzed against distilledwater for 2 days and finally the purified reaction solution islyophilized. The reaction product is a white fibrous solid. TABLE 1Stoichiometric ratios of amounts in the syntheses carried out ProportionMolar ratio Amount of Amount of oxidized NaIO₄:dextran dextranaldehydeof NaIO₄ glucose units Name unit solution solution (%) DA 3 3:5 300 ml 460 ml 30 180 mmol  108 mmol DA 4 4:5 300 ml  612 ml 33 180 mmol  144mmol DA 5 5:5 300 ml  765 ml 49 180 mmol  180 mmol DA 6 2:1 300 ml 1430ml 91 180 mmol  360 mmol DA 8 4:1 150 ml 1430 ml 100   90 mmol  360 mmol

15% strength solutions (w/v) of the dextran-aldehydes prepared in themanner described above were prepared by adding 4.5 g of dextranaldehydeto 30 ml of distilled water and shaking in a waterbath at 37° C.overnight. It is advantageous for the gelation to increase the pH of thedextranaldehyde solution by adding a phosphate buffer.

1.3 Dextran Molecular Weight

The average molecular weight in the dextran was varied. Dextran with anaverage MW of from 60 000 to 90 000 dalton (from Fischer Scientific,Schwerte, Germany) and dextran with a higher average MW of 413 263dalton (from Sigma Aldrich Chemie GmbH, Steinheim, Germany) wasemployed.

The molecular weight had no effect on the proportion of oxidized glucoseunits as a function of the amount of NaIO₄ employed.

Determination of the Aldehyde Content

The percentage content of oxidized glucose units was determined bytitrimetry in analogy to the literature [B. T. Hofreiter, B. H.Alexander, I. A. Wolff, Anal. Chem. 1955, 27, 1930 ff.].

0.15 g of dextranaldehyde is introduced into an Erlenmeyer flask andthen mixed with 10 ml of a 0.25 N carbonate-free NaOH solution. Themixture is stirred until the dextranaldehyde employed is dissolved. Theflask is then immersed in a hot waterbath (80° C.) for one minute andsubsequently placed in an ice bath with vigorous stirring. After oneminute, 15 ml of 0.25 N sulfuric acid are cautiously added whilestirring. The mixture is subsequently diluted with 50 ml of water, and 1ml of 0.2% strength phenolphthalein solution is added. The acidicsolution is titrated with 0.25 N NaOH solution against the indicator.

The dialdehyde content X is calculated from the added amount of dextranor dextranaldehyde and the consumption of acid and base as follows:$X = {\left\lbrack {\frac{\left( {n_{eqbase} - n_{eqacid}} \right)_{DA}}{\frac{W_{DA}}{161}} - \frac{\left( {n_{eqbase} - n_{eqacid}} \right)_{dextran}}{\frac{W_{dextran}}{162}}} \right\rbrack \times 100\%}$

-   X: dialdehyde content-   n_(eqacid): equivalent amount of substance of the acid-   n_(eqbase): equivalent amount of substance of the base-   W_(DA): dry weight of dextranaldehyde-   W_(dextran): dry weight of dextran-   n_(NaOH): normality of the NaOH titer-   n_(H2SO4): normality of the H₂SO₄ solution used

The optimal stoichiometric ratio of NaIO₄ per glucose unit of dextranwas found in further synthesis mixtures. The following graph shows thatthe percentage content of oxidized glucose units is above 90% when thestoichiometric ratio of NaIO₄ per glucose unit of dextran exceeds 2:1.

2. Gelation Time of the Two Solutions

The gelation time depends on the dextranaldehyde used and on the mixingratio of the dextranaldehyde solution and the chitosan solution. Thegelation time increases with an increasing degree of oxidation of thedextranaldehyde and with an increasing 15% strength dextranaldehydesolution:2% strength chitosan solution ratio. It is between 15 and 200seconds. TABLE 2 Gelation times as a function of the dextranaldehydeused and of the mixing ratio of the solutions 2% strength chitosan/15%strength dextranaldehyde solution ratio (ml) Dextranaldehyde 0.5/1.51.0/1.0 DA 3 115 ± 31 s  340 ± 56 s DA 4 64 ± 10 s 194 ± 54 s DA 5  15 ±2.9 s  78 ± 33 s DA 6 19 ± 2 s  15 ± 2 s3. Determination of the Adhesive Shear Force

The adhesive shear force of the novel tissue adhesive is determined withthe aid of purified and lyophilized collagen type I from bovinepericardia (Lyoplant, BBraun Aesculap, Tuttlingen). For this purpose,the Lyoplant is cut into strips 40 mm long and 10 mm wide, with the 1cm² area to be glued being marked at the end thereof. The gluing of theLyoplant strips proceeds as follows:

The appropriate ratios of amounts (see table) of chitosan solution anddextranaldehyde solution are combined in a test tube and shaken for 2seconds in order to obtain thorough mixing of the solutions.Subsequently, 20 μl portions are applied centrally to the area to beglued. The glued area is protected from drying out with a film and isloaded with 50 g for 10 minutes. The strips are then drawn apart at apulling speed of 100 mm/min. The experiments were carried out with twodifferent batches of dextranaldehyde and the average was found for n=13experiments. The results of the experiments are listed in table 3 to 5.TABLE 3 Comparison of the adhesive shear force of DA 3 batch 1 and 2 asa function of the 2% chitosan solution: 15% DA(3) solution mixing ratio2% chitosan solution/15% DA(3) DA 3 adhesive shear DA 3 adhesive shearsolution ratio by force force volume Batch 1 (kPa) Batch 2 (kPa) 3:1 121± 27.6 110 ± 27.6 1:1 167 ± 34.4 154 ± 25.7 1:3 137 ± 38.8 153 ± 33.5

TABLE 4 Comparison of the adhesive shear force of DA 4 batch 1 and 2 asa function of the 2% chitosan solution: 15% DA(4) solution mixing ratio2% chitosan solution/15% DA(4) DA 4 adhesive shear DA 4 adhesive shearsolution ratio by force force volume Batch 1 (kPa) Batch 2 (kPa) 3:1 128± 56   163 ± 56.8 1:1 124 ± 36.4 175 ± 22.2 1:3 167 ± 54.1 192 ± 71.8

TABLE 5 Comparison of the adhesive shear force of DA 5 batch 1 and 2 asa function of the 2% chitosan solution: 15% DA(5) solution mixing ratio2% chitosan solution/15% DA(5) DA 5 adhesive shear DA 5 adhesive shearsolution ratio by force force volume Batch 1 (kPa) Batch 2 (kPa) 3:1 136± 38.7  159 ± 37.1 1:1 148 ± 47.2  187 ± 42.9 1:3 223 ± 46   20.6 ± 41.2

The adhesive shear force increases just like the gelation time withincreasing degree of oxidation and increasing amount of dextranaldehydesolution.

Investigations with dextranaldehyde and a polyvinyl alcohol/vinylaminegraft copolymer (PVALNH₂) were carried out in analogy to thedetermination of the adhesive shear force of thedextranaldehyde/chitosan mixture. The graft copolymer was supplied bythe manufacturer as a 20% aqueous solution and was employed in theundiluted state for gluing Lyoplant strips. The preparation of theLyoplant strips and the application of the solutions were carried outidentically to the dextranaldehyde/chitosan gluings.

The results of the investigations are listed in the tables below: TABLE6 Comparison of the adhesive shear force of DA 4 batch 3 as a functionof the 20% PVALNH₂ solution to 15% DA(4) solution mixing ratio (20%)PVALNH₂ solution/15% DA(4) solution ratio by volume Adhesive shear force(kPa) 3:1 155 ± 25.9 1:1 138 ± 29.0 1:3 159 ± 30.6

TABLE 7 Comparison of the adhesive shear force of DA 5 batch 5 as afunction of the 20% PVALNH₂ solution to 15% DA(5) solution mixing ratioPVALNH₂ solution/15% DA(5) solution ratio by volume Adhesive shear force(kPa) 3:1 145 ± 34.3 1:1 130 ± 19.0 1:3 198 ± 67.4

Additional adhesive shear force investigations were carried out withhigher molecular weight dextranaldehyde and 4% Protasan solution. Thesolutions were applied to the Lyoplant strips with the aid of a Mixpacapplicator. The applicator consists of a two-chamber system with fittedmixer tip. The Lyoplant was cut into strips with a length of 40 mm and awidth of 10 mm, at the end of which a 1 cm² area to be glued was marked.The solutions were applied through the mixer, the glued area was coveredwith a film in order to protect it from drying out and was loaded with50 g for 10 minutes. The strips were then drawn apart at a pulling speedof 100 mm/min. The average MW of the dextran used influences theadhesive shear force, as shown in table 8: TABLE 8 Comparison of theadhesive shear forces of the novel adhesive as a function of the averagemolecular weight of the dextran- aldehyde. 1:1 mixing ratio of thesolutions Adhesive shear DA used Chitosan solution force [kPa] DA 6 oflow average MW 4% Protasan solution 188 ± 38 (15% solution) DA 6 of highaverage MW 4% Protasan solution 278 ± 71 (15% solution)

Adhesion tests were likewise carried out with Bioglue® (CryolifeInternational Inc. USA) consisting of proteins and glutaraldehyde, andwith GLUETISS a gelatin-resorcinol dialdehyde adhesive, which wereapplied to the Lyoplant strips in accordance with the instructions foruse. The strips glued with these adhesives were likewise covered with afilm and loaded with 50 g for 10 minutes. A comparison of the adhesiveshear forces achieved is shown in table 9. TABLE 9 Comparison of theadhesive shear force of a composition of the invention with BioGlue ®and GLUETISS ® Adhesive shear Mixing force Adhesive Composition ratio[kPa] Composition of 4% Protosan CI 213 1/4 245 ± 68 the invention and15% DA 6 solution Composition of 4% Protosan CI 213 1/1 278 ± 71 theinvention and 15% DA 6 solution Composition of 4% Protosan CI 213 2/1262 ± 78 the invention and 15% DA 6 solution Bioglue Albumin solution/4/1 178 ± 54 glutaraldehyde solution Gluetiss Gelatin-resorcinol as 167± 37 solution/dialdehyde directed solution4. Stoppage of Liver Bleeding

The surgical glue was employed to stop bleeding in the rat liver (SPFWistar rats). A composition of a 4% strength aqueous Protasan solutionand of a 15% strength aqueous dextranaldeyde solution DA 6 was chosenfor this purpose. The solutions were employed in a mixing ratio of 1:1.A Mixpac applicator was used to mix and apply the components.

After the rats had been anesthetized, the model of a crosswise incision(length of the cuts: 2.5 cm) on the liver was chosen. The adhesive wasapplied to the bleeding wound immediately after the incision. A gelformed and adhered firmly to the liver surface, so that the bleedingceased immediately after application of the adhesive.

1. A composition of at least two, in particular two, biocompatiblecomponents which can be chemically crosslinked together, in particularfor gluing biological tissue, comprising at least the followingcomponents: a) aqueous solution of at least one polymer having aminogroups b) aqueous solution of at least one aldehyde having at leastthree aldehyde groups, where the stoichiometric amount of aldehydegroups in component b) is at least three times the stoichiometric amountof amino groups in the polymer having amino groups, and the compositionis free of protein.
 2. The composition as claimed in claim 1,characterized in that the aldehyde and the polymer having amino groupscan be crosslinked together at body temperature.
 3. The composition asclaimed in either of claims 1 or 2, characterized in that the polymerhaving amino groups is derived from a biodegradable natural material. 4.The composition as claimed in any of the preceding claims, characterizedin that the polymer having amino groups is a polysaccharide, inparticular a modified polysaccharide, in which the amino groups areliberated by deacetylation.
 5. The composition as claimed in any of thepreceding claims, characterized in that the polymer having amino groupsis chitosan.
 6. The composition as claimed in any of the precedingclaims, characterized in that the polymer having amino groups is an atleast partially deacetylated chitin having a degree of deacetylation offrom 50 to 100%, preferably 60 to 90%, in particular 70 to 80%.
 7. Thecomposition as claimed in any of the preceding claims, in particular asclaimed in claim 1 or 2, characterized in that the polymer having aminogroups is a synthetic polymer, in particular a polymer which undergoesrenal elimination.
 8. The composition as claimed in claim 7,characterized in that the synthetic polymer is a modified polyvinylalcohol having amino groups, preferably having a molecular weight of ≦50000, in particular ≦20
 000. 9. The composition as claimed in any of thepreceding claims, characterized in that the aldehyde is a polyaldehyde.10. The composition as claimed in any of the preceding claims,characterized in that the aldehyde is an oxidized polysaccharide. 11.The composition as claimed in claim 10, characterized in that thepolysaccharide is at least one from the group of dextran, chitin,starch, agar, cellulose, alginic acid, glycosaminoglycans, hyaluronicacid, chondroitin sulfate and derivatives thereof.
 12. The compositionas claimed in any of the preceding claims, characterized in that thealdehyde, in particular dextranaldehyde, has a molecular weight of about60 000 to 600 000, in particular about 200
 000. 13. The composition asclaimed in any of the preceding claims, characterized in that thealdehyde is partially or completely masked.
 14. The composition asclaimed in claim 13, characterized in that the aldehyde is masked withan S, O or N nucleophile.
 15. The composition as claimed in either ofclaims 13 or 14, characterized in that the partially or completelymasked aldehyde is a polysaccharide-alkali metal bisulfite adduct. 16.The composition as claimed in either of claims 13 or 14, characterizedin that the aldehyde is partially or completely masked with ethanol orglycerol.
 17. The composition as claimed in any of the preceding claims,characterized in that the content of aldehyde of component b) is from 5to 20% by weight, in particular from 10 to 15% by weight.
 18. Thecomposition as claimed in any of the preceding claims, characterized inthat the content of polymer having amino groups of component a) is from1 to 25% by weight, in particular from 2 to 20% by weight.
 19. Thecomposition as claimed in any of the preceding claims, characterized inthat the ph values of the components are adjusted so that the ph of amixture of the components is between 3 and 8, in particular between 5and 7.5.
 20. The composition as claimed in any of the preceding claims,characterized in that the components are adjusted with respect to oneanother so that they crosslink together in a short time, in particular atime of from 15 to 200 sec, after they are combined.
 21. The compositionas claimed in any of the preceding claims, characterized in that theviscosities of the components are adjusted in relation to one another sothat a layer of the composition with a thickness of from 0.1 to 1 mm canbe applied.
 22. The composition as claimed in any of claims 1 to 6 and 9to 18, characterized in that component a) is a solution of chitosan inacetic acid, and component b) is an aqueous solution of dextranaldehyde.23. The provision of the composition as claimed in any of the precedingclaims for use as surgical tissue adhesive, in particular for sealing orclosing surfaces or orifices.
 24. The use as set forth in claim 23,characterized in that the components are mixed together shortly beforeapplication.
 25. The use as set forth in claim 23, characterized in thatthe components are mixed on an application site.
 26. A kit consisting oftwo substantially separate containers, where the containers each containone component of the composition as claimed in any of claims 1 to 22.27. The kit as claimed in claim 26, characterized in that the twocontainers function as syringe barrels of a double syringe.
 28. The kitas claimed in either of claims 26 to 27, characterized in that it has adevice for mixing the components.
 29. The kit as claimed in any ofclaims 26 to 28, characterized in that it has a static mixer which canbe fitted in particular onto a double syringe.